Glaucoma is a neurodegenerative disorder that is the leading cause of blindness in the world. While current treatments are aimed toward reducing the intraocular pressure (lOP), neurodegeneration sometimes continues even with a reduction in lOP. One of the mechanisms that may lead to the selective destruction of retinal ganglion cells is a glutamate-mediated excitotoxicity. Unfortunately, current methods for measuring glutamate in eye tissues do not provide all The desired spatial resolution for characterizing retinal glutamate levels and their potential sources. The goal of this proposal is to develop and demonstrate a novel, in vivo sampling system that provides the needed spatial localization for characterizing vitreal glutamate levels in a rat model of glaucoma. In the first phase of this project, a miniaturized, low-flow push-pull perfusion method and an appropriate capillary electrophoresis assay for glutamate that have been developed in our lab will be adapted for use in the vitreous. The spatial resolution of our low-flow push-pull perfusion will be used to characterize glutamate levels at sites in the central vitreous and vitreoretinal interface. Additionally, the source and dynamics of glutamate levels at the probe tip will be assessed by the inclusion of pharmacological agents into the perfusion solution. In the second phase of this project, the sampling method will be used to address the hypothesis that glutamate levels increase in specific locations at the retina in response to a chronic, moderate increase in lOP in a rat model of glaucoma. Measurements of vitreoretinal glutamate levels will be performed at central and peripheral sites of the retina during the first two weeks of a moderate increase in lOP. The outcomes of this project will be the introduction of a new tool for in vivo sampling in eye tissues and will provide previously inaccessible information regarding the neurobiology of glutamate at the glaucomatous retina. Further, the ability to characterize chemical composition at the retina demonstrated in this study may be applicable to innovative studies of other diseases of the retina.